Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians, with an incidence of one bird in 2500-4000 and 70,000 affected people worldwide and kills most of them in their 20s. Cystic fibrosis is an autosomal recessive disease linked to mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene whose nature determines the clinical expression and severity of the disease, affecting mainly the respiratory, digestive and genital systems. CFTR, a chloride-ion channel, is involved in the changes of surface liquid covering airway epithelial cells. Dehydration of the surface liquid leads to altered mucociliary clearance and inflammation and infections at the mucosal epithelia. CFTR mutations that reduce CFTR protein function cause accumulation of thick, sticky mucus in the bronchi of the lungs, loss of exocrine pancreatic function, impaired intestinal secretion, and an increase in the concentration of chloride in the sweat (Boucher R C Trends Mol Med., 2007). In patients with CF, lack of CFTR Cl(−) channel function leads to progressive pulmonary damage and ultimately to death. Chronic lung disease is the major cause of mortality and morbidity in CF patients.
Patients with CF require numerous therapies to manage these symptoms (Farrell P M et al. J Pediatr, 2008), including mucolytic and antibiotic agents and chest physiotherapy to treat the airway disease and digestive enzymes to replace the loss of exocrine pancreatic function. These and other interventions have increased life expectancy dramatically, but improvement is needed to reduce the high treatment burden and increase survival (Sawicki G S et al. J Cyst Fibros, 2009). A CFTR potentiator (Ivacaftor) has been developed by Vertex Pharmaceuticals (Ramsey B W et al. N Engl J Med, 2011) and has been recently approved for the treatment of CF patients carrying the p.Gly551Asp mutation (2-5% of all patients). To date, the drug has failed for CF patients with p.Phe508del, the most common mutations. Several CFTR correctors have been previously reported to be active in vitro (Hutt D M et al. Nature Chem Biol, 2010; Verkman A S and Galietta L J Nat Rev Drug Discov, 2009) but therapies for CF have not yet advanced from these efforts. A corrector that corrects the trafficking of the p.Phe508del protein is still under investigation in clinical trials (Van Goor F et al. PNAS, 2011).
Accordingly, there is a need to develop new drugs that will be suitable for preventing or treating CF and CFTR-related diseases. In this way, it has been suggested that characterization of new therapeutic compounds in CF and CFTR-related diseases may be highly desirable.
Since the cloning of the CFTR gene in 1989, nearly 2000 mutations of the gene have been described. The p.Phe508del mutation (deletion of phenylalanine at position 508 of the protein) is the most frequent (70% of mutated alleles in patients with CF). This severe mutation impairs the maturation of CFTR protein and the protein fails to reach cell membrane. Other grouped according to their effect on the CFTR protein include mutations that result in a shortened protein, in a reduced chloride conductance, in a defective CFTR stability at the cell surface or in reduced numbers of CFTR transcripts due to incorrect splicing (Rowe S M et al. N Engl J Med, 2005).
Alternative splicing is a regulated process during gene expression that results in a single gene coding for multiple proteins, constituting important post-transcriptional regulation of eukaryotic gene expression. In this process, particular exons of a gene may be included within, or excluded from, the final, processed mRNA. Abnormal variations in splicing are also implicated in disease; a large proportion of human genetic disorders result from splicing variants. Different CFTR mutations in splice sites or cis-acting splicing regulatory sites or resulting in the creation of new abnormal alternative donor or acceptor site may lead to mis-splicing of multiple abnormal CFTR transcripts and non functional CFTR proteins.
In addition to alternative splicing mechanisms, microRNAs (miRNA) can act in synchrony with transcription factors to control gene expression (Martinez N J et al. Bioessays, 2009; Shalgy R et al. Aging, 2009), revealing an important new complexity in the post-transcriptional regulation of eukaryotic gene expression. Recently, it has been found that CFTR is post-transcriptionally regulated by miRNAs, such as miR-145 and miR-494 (Gillen A E et al. Biochem J, 2011; Megiorni F et al. Plos One, 2011; Ramachandran S et al. PNAS, 2012). Several miRNAs including miR-145 are expressed in primary human airway epithelial cells, where CFTR expression is repressed (Gillen A E et al. Biochem J, 2011) or are deregulated in CF patients (Oglesby I K et al. J immunol, 2013; Ramachandran S et al. AJRCMB, 2013).